Two new species of Meliola from Yunnan Province China

683 MycoKeys

MycoKeys 121: 79-92 (2025)
DOI: 10.3897/mycokeys.121.158055

Research Article

Two new species of Meliola from Yunnan Province China

Muhammad Binyamin Khan'23®, Xiang-Yu Zeng*®, Entaj Tarafder*®, Sardar Ali?°*®, DE-Ping Wei'®,

Ting Chi Wen'234®

1 State Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University,

Guiyang 550025, China

2 Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, Guiyang 550025, China
3 School of Pharmacy, Guizhou University, Guiyang 550025, China

4 Department of Plant Pathology, College of Agriculture, Guizhou University, Guiyang, China

Corresponding author: Ting Chi Wen (tingchiwen@yahoo.com)

OPEN Qaceess

Academic editor: Rajesh Jeewon
Received: 6 May 2025

Accepted: 24 July 2025

Published: 21 August 2025

Citation: Khan MB, Zeng X-Y, Tarafder
E, Ali S, Wei D-P Wen TC (2025)

Two new species of Meliola from
Yunnan Province China. MycoKeys
121: 79-92. https://doi.org/10.3897/
mycokeys.121.158055

Copyright: © Muhammad Binyamin Khan et al.
This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International - CC BY 4.0).

Abstract

Meliola is an epifoliar fungal genus commonly known as black mildews, predominantly
found in tropical and subtropical regions and considered host specific. This study de-
scribes two novel species, Meliola fuscobrunnea and M. fusconigra, collected from Yunnan
Province, China. Meliola fuscobrunnea was collected from the leaves of Annona squamosa,
while M. fusconigra was collected from Xylopia aethiopica. Comprehensive morpho-mo-
lecular analyses were conducted to distinguish these taxa from other Meliola species. Phy-
logenetic analyses based on small subunit (SSU) and large subunit (LSU) rDNA sequences
confirmed their placements within Meliola and supported their status as distinct lineages.
The morpho-molecular data provide robust evidence to establish these species as novel
taxa, enriching the current understanding of fungal diversity in Yunnan Province.

Key words: Black mildews, fungal diversity, new species, phylogenetic analysis,
taxonomy

Introduction

Meliola was introduced by Fries (1825), and later, Martin (1941) established
Meliolaceae based on Meliola as the generic type (Hawksworth and Eriks-
son 1986; Hongsanan et al. 2015; Hyde et al. 2024). Lumbsch and Huhndorf
(2010) placed Meliolales in Sordariomycetes. Kirk et al. (2008) reported over
1,200 species of Meliola, and the genus presently contains 2,365 species
and 701 varieties (Index Fungorum 2025, accessed April 2025). Meliola is
one of the most important genera and usually occurs on leaves, petioles,
twigs, and fruits (Hosagoudar 2003; Zeng et al. 2017; Jayawardena et al.
2020; Santos et al. 2021; Bermudez Cova et al. 2022; Zeng et al. 2022; Khan
et al. 2025). Despite its morpho-species richness, Meliola faces fundamen-
tal classification challenges, primarily due to the traditional reliance on host
specificity for species delimitation (Zeng et al. 2019, 2020). This practice
has led to the description of numerous species based solely on host associ-
ation, often without adequate morphological or molecular evidence (Justavi-
no and Piepenbring 2007; Marasinghe et al. 2020).

19

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

However, host specificity cannot be accepted as a reliable taxonomic crite-
rion, as certain Meliola species may occur on multiple hosts, while morpholog-
ically similar but genetically distinct species may co-occur on the same host
(Santos et al. 2021; Khan et al. 2025). Morphologically, Meliola species are
characterized by superficial black colonies; globose to subglobose ascomata
with superficial mycelia; 2—4-spored, unitunicate asci; and 3—4-septate brown
ascospores (Pinho et al. 2012a; Hongsanan et al. 2015; Justavino et al. 2015;
Hyde et al. 2020a, 2020b). According to phylogenetic analyses based on LSU
and ITS sequences, the placement of Meliola has been confirmed in Sordario-
mycetes, although it appears to be polyphyletic due to limited molecular data
(Gregory and John 1999; Pinho et al. 2012a, 2012b; Hongsanan et al. 2015;
Justavino et al. 2015; Hyde et al. 2020b; Marasinghe et al. 2020; Zeng et al.
2020). The species of Meliola are usually biotrophic and difficult to culture us-
ing artificial media. Therefore, direct DNA extraction from fruiting structures is
often susceptible to contamination by other fungal species. These persistent
issues underscore the urgent need for a comprehensive taxonomic revision of
Meliola, incorporating detailed morphological examination and multilocus mo-
lecular phylogenetic analyses (Hongsanan et al. 2015; Zeng et al. 2017).

This study introduces Meliola fuscobrunnea and M. fusconigra as new spe-
cies collected from Annona squamosa and Xylopia aethiopica, respectively, in
Yunnan Province, China. Xylopia aethiopica (Dunal) A. Rich. has previously been
recorded as a host for Meliola species (M. xylopiae F. Stevens and M. kuprensis
Deighton) (Deighton 1951; Hansford 1961). In contrast, Annona squamosa L. is
reported here for the first time as a host for the genus Meliola. Comprehensive
morpho-molecular analyses supported their distinct placements within Meli-
ola. These findings contribute to the taxonomy of Meliola, improve understand-
ing of host-specific plant-fungus interactions, and enrich fungal biodiversity
records in an underexplored region of Yunnan Province, China. Moreover, the
results provide essential baseline data for future ecological, biogeographical,
and evolutionary studies and support global initiatives aimed at documenting
fungal diversity in the context of environmental changes.

Materials and methods
Study area

A field survey was conducted in July 2024 in Yunnan Province, China, a global bio-
diversity hotspot known for its rich fungal diversity, accounting for over 40% of all
fungal species recorded in China (Feng and Yang 2018; Liu et al. 2018; Phookam-
sak et al. 2024). The study focused specifically on the forests of Yunnan Prov-
ince, China. This region receives an annual rainfall of 1000-4000 mm and has a
distinct seasonal climate, with cold and dry conditions from November to April
and warm, humid conditions from May to October (Dumbacher et al. 2011).

Morphological characterization

For the morphological observations, infected plant leaves were first examined
using a stereomicroscope (LABOMED America, 7GA9, USA). A small amount
of fungal material was gently scraped from the leaf surface using a fine needle

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Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

and mounted in a drop of 5% aqueous KOH on a clean glass slide for obser-
vation using a compound microscope (Senanayake et al. 2020). Microscopic
features of the mycelium, conidiophores, conidia, and other hyphal characters
were examined at magnifications of 4x, 10x, and 40x. Measurements were
made with an ocular micrometer at 40x. Light micrographs of microscopic fea-
tures of the fungal pathogen were also captured.

DNA extraction, amplification, and sequencing

Genomic DNA was directly extracted from the isolated fruiting structures of
M. fuscobrunnea and M. fusconigra scraped from the infected leaf surfaces
of A. squamosa and X. aethiopica, respectively, using the E.Z.N.A. fungal DNA
kit (Omega Bio-Tek, Norcross, GA, USA). The procedures followed the manu-
facturer's protocol, and the extracted DNA was stored at -20 °C. Partial se-
quences from the SSU region of the nrDNA were amplified under standard PCR
conditions using the primer pair NS1/NS4 (White et al. 1990). The LSU region
was amplified using the primer pair LROR/LR5 (Vilgalys and Hester 1990). A
25 uL reaction mixture containing 1.6 uL dNTP mix (2.5 mM/mL), 0.2 uL Taq
polymerase (5 U/mL), 2 uL polymerase buffer (10x), 1 pL each of forward and
reverse primers (10 mM/mL), and 1 pL DNA template was used for the PCR
experiments. Amplifications were carried out in a T100™ Thermal Cycler (BIO-
RAD) (Table 1). Sangon Biotech (Shanghai) Co., Ltd. sequenced the PCR prod-
ucts using the same primers used in the amplification reactions.

Phylogenetic analysis

The newly generated forward and reverse sequences were assembled us-
ing BioEdit v. 7.2.5 (Hall 1999) to generate a consensus sequence. A BLAST
search (https://blast.ncbi.nlm.nih.gov) was conducted through the NCBI Gen-
Bank database to identify and retrieve reference sequences homologous to
Meliola sp. The reference sequences were obtained from the literature and
GenBank. The sequences (SSU and LSU) were independently aligned using
the online MUSCLE version (Edgar 2004), and BioEdit was used for manual
editing and refinement. Sordaria fimicola (Roberge ex Desm.) Ces. & De Not.
(CBS 723.96) was selected as the outgroup taxon (Zeng et al. 2022). Max-
imum likelihood (ML) phylogenetic analyses were performed using RAXML
(Miller et al. 2010) with the GTRGAMMA model, and 1,000 bootstrap repli-
cates were used to assess branch support. Phylogenetic analyses were con-
ducted on the CIPRES Science Gateway platform, and the resulting phyloge-
netic tree is presented in Fig. 1. New species are established based on the
recommendations outlined by Jeewon and Hyde (2016).

Table 1. The PCR conditions and the primers used in this study.

Locus Primer

SSU NS1
NS4
LSU LROR

LRS

Sequence PCR condition References
GTAGTCATATGCTTGTTC (1) Initial denaturation for 3 min at 94 °CC White et al. (1990)
CTTCCGTCAACCTTTAAG (2) 40 cycles of denaturation at 94 °C for 45 s, annealing

at 56 °C for 50 s, and extension at 72 °C for 1 min
ACCCGCTGAACTTTAAGC (3) Final elongation at 72 °C for 10 min Vilgalys and Hester (1990)
TCCTGAGGGAAACTTCG (4) Storage at 4 °C

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 81

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

88

frenopsis vincensii - VIC:31752
Appendiculetia viticis - MFLU 19-1008

Asteridiella obesa - VIC:31239
Meliola tamarindi - MFLU 14-0080

lrenopsis walsurae - MFLU130 oe 21

Figure 1. Molecular phylogenetic analysis of Meliola species by the maximum likelihood (ML) method based on com-
bined SSU and LSU sequences. The tree is rooted with Sordaria fimicola (CBS 723.96). Newly generated type sequences

are shown in blue.

Results
Phylogenetic analyses

The newly generated sequences were deposited in GenBank, and their accession
numbers are provided in Table 2. The combined dataset for phylogenetic analysis
incorporated nuclear ribosomal loci (SSU and LSU) representing 42 strains, with
Sordaria fimicola CBS 723.96 (Sordariaceae, Sordariales) designated as the out-
group taxon. The RAXML analysis of the combined dataset yielded the best scor-
ing tree (Fig. 1), with a final ML optimization likelihood value of -18855.431359.

Phylogenetic analyses revealed that our strains were placed within Me-
liola and clustered independently from other species. Meliola fuscobrun-
nea (HKAS 146649, HKAS 146650) forms a strongly supported clade with
M. clerodendri-infortunati X.Y. Zeng, K.D. Hyde and T.C. Wen (MFLU 13-0624,
MFLU 16-0069), with high statistical support (MLBS = 99%), indicating a close
phylogenetic affinity between these taxa. Meliola fusconigra (HKAS 146645,
HKAS 146646, HKAS 146647, and HKAS 146648) formed a well-supported
clade (MLBS = 92%), closely related to Meliola aristolochiae-tagalae X.Y. Zeng,
K.D. Hyde and T.C. Wen (MFLU 160088), with strong support (MLBS = 90%),
suggesting a close evolutionary relationship between the two species (Fig. 1).

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 ry)

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

Table 2. Fungal species used for phylogenetic analyses of Meliola. Newly generated sequences from this study are

aon

shown in bold, while

Species

Amazonia acaciae
Amazonia goosii
Amazonia leeae
Appendiculella sp.
Appendiculella viticis
Asteridiella combreticola
Asteridiella obesa
Endomeliola dingleyae
lrenopsis crotonicola
lrenopsis vincensii
lrenopsis walsurae
Meliola fuscobrunnea
Meliola fuscobrunnea
Meliola fusconigra
Meliola fusconigra
Meliola fusconigra
Meliola fusconigra
Meliola acacia

Meliola aristolochiae-tagalae
Meliola centellae

Meliola citri-maximae
Meliola clerodendricola
Meliola clerodendri-infortunati
Meliola clerodendri-infortunati
Meliola jasmini-sambac
Meliola jasmini-sambac
Meliola lithocarpigena
Meliola pistaciicola
Meliola pottsiae

Meliola tamarindi
Meliola telosmae
Meliola thailandicum
Meliola thailandicum
Meliola thailandicum
Sordaria fimicola

indicates data unavailable.

Genbank accession number

Strain
SSU

VIC32061 KC618656

MTCHT146 OM296101

MTCHT147 OM296099

MP3432 DQ508301

MFLU 19-1008
MFLU 17-1041 MN747498

VIC 31239 KC618653

PDD:98304
MFLU:14-0078 KY554796

VIC:31752
MFLU13-0621 MN747487
HKAS 146650 PV299283
HKAS 146649 PV298266
HKAS 146645 PV298261
HKAS 146646 PV299282
HKAS 146647 PV298262
HKAS 146648 PV298263
MFLU 16-0077 NG_077426
MFLU 160088 MN747496

VIC:31244
MFLU 14-0288 NG_070325
MFLU13-0620 MN747486
MFLU:13-0624 NG_070324
MFLU 16-0069
MFLU 17-1044 MN747499
MFLU 17-1046 MN747500
MFLU 13-0628 NG_077423
MFLU 16-0070 NG_077425
MFLU 13-0631 NG_077424
MFLU:14-0080 KY554797
MFLU 14-0003 MK103390
MFLU 16-0067

MFLUCC:15-0378
MFLUCC:15-0047
CBS 723.26
Taxonomy

LSU

MT108888

NG057014
GU138866

JX133163

PV298265
PV298264
PV298245
PV446596
PV297977
PV454349

NG042650

MN788607
MT911464

MN788606
KR868695
KR868698
MH874231

Country

Brazil
India
India
Panama
Thailand
Thailand
Brazil
New Zealand
Thailand
Brazil
Thailand
China
China
China
China
China
China
Thailand
Thailand
Brazil
Thailand
India
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Thailand
Philippines
Thailand
Thailand
Thailand
Thailand

Papua New Guinea

References

Pinho et al. 2012b
Hosagoudar and Abraham (1998)
Hansford and Thirumalachar (1948)
Rodriguez and Piepenbring (2007)
Marasingheet al. 2020
Zeng et al. 2022
Pinho et al. 2012b
Hughes and Pirozynski (1994)
Zeng et al. 2018
Zeng et al. 2018
Zeng et al. 2018
This study
This study
This study
This study
This study
This study
Zeng et al. 2020
Zeng et al. 2020
Pinho et al. 2012b
Hyde et al. 2017
Minter 2006
Zeng et al. 2017
Zeng et al. 2017
Zeng et al. 2020
Zeng et al. 2020
Zeng et al. 2020
Zeng et al. 2020
Zeng et al. 2020
Zeng et al. 2017
Zeng et al. 2020
Hongsanan et al. 2015
Hongsanan et al. 2015
Hongsanan et al. 2015
Zeng et al. 2020

Meliola fuscobrunnea Khan M.B. & T. C. Wen, sp. nov.
MycoBank No: 858334

Fig. 2

Etymology. From Latin, “fusco” meaning “dark,” and “brunnea” meaning “brown,”
referring’ to the distinct dark brown color of Ascospores.

Diagnosis. Appressoria small and variable in size and slightly thick; asco-
mata superficial and relatively small; setae moderately long, straight to slightly
curved with well-developed, clearly visible phialides; ascospores cylindrical or
oblong and comparatively large, and dark brown in color.

Holotype. CHINA * Yunnan Province, Gaoligong Mountains, Muhammad
Binyamin Khan, 27 July 2024, HKAS 146650, GenBank accession numbers
PV299283 (SSU) and PV298265 (LSU).

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055

83

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

H | J

Figure 2. Meliola fuscobrunnea A. Host leaves; B. Colonies on the leaf surface; C. Ascomata on leaf surface; D. Phialides;
E, F. Reticulate superficial hyphae with appressoria; G. Hyphal setae; H, I. Asci; J. Ascospore; K. Immature asci. Scale
bars: 1 mm (B); 10 (D, E); 20 um (F-K).

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 84

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

Additional materials examined. CHINA * Yunnan Province, Gejia Mountain,
Muhammad Binyamin Khan, 26 July 2024, HKAS 146649, GenBank accession
numbers PV298266 (SSU) and PV298264 (LSU).

Habitat. On leaving leaves of Annona squamosa.

Description. Biotrophic on the surface of living leaves of A. squamosa. Colonies
5-10 mm in diameter, epiphyllous, scattered, black. Hyphae superficial, black,
straight to substraight, branched, septate, each cell 19-21 um long (X = 20 um,
n = 20), reticulate with dark brown setae. Hyphal setae up to 305 um long, subdense,
dark brown, straight to curved, 2 dentate, obtuse. Appressoria 8-18 x 4-11 um
(x= 13 x 7 um, n = 30), 2 celled, brown, clavate, straight to curved, formed near
the septa, unilateral, antrorse. Sexual morph: Ascomata up to 200 um in diameter,
superficial, subdense, dark, globose to subglobose, with a central ostiole. Peridium
comprises hyaline inner cell and dark brown outer wall with textura angularis. Ha-
mathecium with evanescent paraphyses. Ascospores 46-52 x 14-18 um (x= 49
x 16 um, n = 20), 2-3 seriate, cylindrical or oblong, hyaline when young, becoming
dark brown when mature, 4 septate, constricted at the septa, rounded at both ends,
smooth walled. Asexual morph: Phialides 17-25 x 5-9 um (X= 21 x 77 um, n= 10),
opposite to unilateral, flask-shaped, mixed with appressoria, ampulliform.

Notes. Meliola fuscobrunnea (HKAS 146649, HKAS 146650) forms a strong-
ly supported clade with M. clerodendri-infortunati (MFLU 13-0624, MFLU 16-
0069) with high statistical support (MLBS 99%) (Fig. 1). Both species have
similar orientation and arrangement of superficial hyphae, hyphal setae length,
appressorium colour and position, ascomata shape, and peridium wall colour.
Meliola clerodendri-infortunati is distinct from M. fuscobrunnea in hyphal setae
apex and size of appressorium, ascomata, and ascospore. Additionally, the two
species also differ in host association, with the former found on Clerodendrum
infortunatum L. and the latter on A. squamosa (Hyde et al. 2017). Hence, based
on the differences in morphological characteristics, phylogenetic analyses, and
host differences, we introduce M. fuscobrunnea as a new species of Meliola.

Meliola fusconigra Khan M.B. & T. C. Wen, sp. nov.
MycoBank No: 858335
Fig. 3

Etymology. From Latin, “fusco” meaning dark and “nigra” meaning “black,” refer-
ring to the distinct dark black colonies of species on natural substrate.

Diagnosis. Appressoria clavate, brown, unilateral, and antrorse; ascomata
globose to subglobose with a central ostiole. Phialides flask-shaped, well-de-
veloped, and mixed with appressoria; ascospores cylindrical to oblong, large,
smooth-walled, and dark brown at maturity.

Holotype. CHINA * Yunnan Province, Laifengshan National Forest Park, Mu-
hammad Binyamin Khan, 26 July 2024, HKAS 146645, GenBank accession
numbers PV298261 (SSU) and PV298245 (LSU).

Additional materials examined. CHINA * Yunnan Province, Gaoligongshan,
Muhammad Binyamin Khan, 27 July 2024, HKAS 146646, HKAS 146647,
and HKAS 146648, GenBank accession numbers PV299282, PV298262, and
PV298263 (SSU); PV446596, PV297977, and PV454349 (LSU)

Habitat. On leaving leaves of Xylopia aethiopica.

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 85

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

Figure 3. Meliola fusconigra A. Host leaves; B, C. Ascomata colony on surface of leaves; D, E. Reticulate hyphae with
appressoria; F. Hyphal setae; G-I. Ascospores. Scale bars: 1 mm (B, C); 20 um (D-I).

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 86

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

Description. Biotrophic on the surface of living leaves of X. aethiopica. Colo-
nies 4-8 mm in diameter, epiphyllous, dense to subdense, dark black. Hyphae
superficial, black, straight to substraight, branched, black dark at septa, each
cell 18-88 um long (x = 53 um, n = 10), loosely reticulate, with dark black setae.
Hyphal setae 291-306 x 2-6 um (X= 298 x 4 um, n = 10), narrowly cylindrical,
rounded to acute at the apex. Appressoria 24—28 x 11-15 um (X= 26 x 13 um,
n = 20), 2 celled, brown, clavate, substraight, formed near the septa, unilater-
al, antrorse. Ascomata up to 250 um in diameter, superficial, subdense, dark
brown, globose to subglobose, with a central ostiole. Peridium comprises hya-
line inner cell and dark brown outer wall with textura angularis. Hamathecium
with evanescent paraphyses. Ascospores 48-60 x 15-21 um (xX = 54 x 18 um,
n = 20), 3-4 seriate, cylindrical or oblong, hyaline when young, becoming dark
brown when mature, 4 septate, constricted at the septa, rounded at both ends,
smooth walled. Asexual morph: Phialides 40-30 x 5-9 um (Xx = 35 x 7 um,
n = 10), opposite to unilateral, sometimes alternate, flask-shaped, few mixed
with appressoria, ampulliform.

Notes. Phylogenetic analyses showed that M. fusconigra forms a sister
clade to M. aristolochiae-tagalae with strong statistical support (MLBS = 90%),
confirming their close evolutionary relationship (Fig. 1). Both species share
similarity in colony and peridium cell colour, orientation of appressoria, and
shape of appressoria, ascomata, and ascospore (Zeng et al. 2022). However,
notable differences can be observed between M. fusconigra and M. aristolochi-
ae-tagalae in the colour of superficial hyphae and the size of individual cells of
superficial hyphae, appressoria, ascomata, ascospores, and phialides, and host
species (Zeng et al. 2022). This morpho-anatomical comparison with the phy-
logenetically allied species represents M. fusconigra as a distinct new species.

Discussion

This study proposes two new species, Meliola fuscobrunnea and M. fuscon-
igra. These species were collected from the mountains of Yunnan Province,
China, and were identified as novel taxa based on morpho-molecular evidence.
Annona squamosa is reported here for the first time as a host for the genus
Meliola. Meliola fuscobrunnea (HKAS 146649, HKAS 146650) forms a strongly
supported clade with M. clerodendri-infortunati (MFLU 13-0624, MFLU 16-0069)
with high statistical support (MLBS = 99%) and differs from M. clerodendri-in-
fortunati (MFLU 16-0069) by 70 nucleotide substitutions in the LSU region and
by 9 substitutions in the SSU region when compared with the SSU sequence of
the same species (NG_070324) (Jeewon and Hyde 2016). Both species have
straight to substraight and reticulated superficial hyphae, up to 300 um long
and dark brown hyphal setae, brown and unilateral to antrorse appressorium,
globose to subglobose ascomata, and a hyaline inner and dark brown outer
wall of peridium. However, the former can be distinguished from the latter by
its hyphal setae with acute apex, slightly larger appressoria (14 x 9 pm), slightly
smaller ascomata (up to 160 um diam.), smaller ascospores (39 x 9 um), and
rarity of phialides. Moreover, M. clerodendri-infortunati has only cylindrical as-
cospores, while M. fuscobrunnea has oblong ascospores in addition to cylindri-
cal ascospores. Meliola clerodendri-infortunati was recovered from the living
leaves of C. infortunatum (Hyde et al. 2017), while Meliola fuscobrunnea was

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 87

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

found on the living leaves of Annona squamosa in the present study, indicating
that the two species might differ in their host association.

Meliola fusconigra (HKAS 146645, HKAS 146646, HKAS 146647, and HKAS
146648) formed a well-supported clade (MLBS = 92%), indicating its distinct
phylogenetic placement. It is closely related to Meliola aristolochiae-tagalae
(MFLU 160088), with strong bootstrap support (MLBS = 90%), suggesting a
close evolutionary relationship between the two species. It differs from Meli-
ola aristolochiae-tagalae (MFLU 160088) by 20 nucleotide substitutions in the
SSU region; however, LSU sequence data for M. aristolochiae-tagalae were not
included in this study (Jeewon and Hyde 2016). Meliola aristolochiae-tagalae
shares common morphological features with M. fusconigra, such as colony
color (black), orientation of superficial hyphae (straight to substraight), ori-
entation and shape of appressoria (unilateral and clavate), ascomata shape
(globose to subglobose), peridium cell color (hyaline inner cell and dark brown
outer wall), and ascospore shape (cylindrical or oblong). However, M. aristo-
lochiae-tagalae differs from M. fusconigra in having brown, closely reticulated
superficial hyphae with relatively smaller individual cells (19 um long), rela-
tively larger (up to 420 um), dark brown, and straight hyphal setae, shorter
appressoria (17 um), smaller ascomata (200 um diam.), smaller ascospores
(42 x 14 um), and 2-3-seriate ascospores, and smaller, unilateral phialides
(18 x 8 um), while M. fusconigra contains brown and loosely reticulated super-
ficial hyphae with larger individual cells (53 um long), smaller (298 um), black,
and narrowly cylindrical hyphal setae, longer appressoria (26 um), bigger as-
comata (250 um diam.), bigger ascospores (54 x 18 um), and 3-4-seriate
ascospores, and bigger, opposite to unilateral, sometimes alternate phialides
(35 x 7 um). Moreover, both species have different hosts, as the former was
reported to infect Aristolochia tagala Cham., while the latter was found in as-
sociation with X. aethiopica (Zeng et al. 2022).

This study identified Meliola fuscobrunnea and M. fusconigra as new spe-
cies, supported by comprehensive morpho-molecular analyses. Moreover, this
study also expands the known host range of Meliola by recording Annona squa-
mosa as anew host while contributing to the understanding of fungal diversity
in Yunnan Province, China.

Acknowledgments

We are thankful to Prof. Dr. Munazza Kiran, Department of Botany, University of
Education, Lahore, Pakistan, for critically reviewing the manuscript. Additional-
ly, we extend our sincere thanks to all individuals and organizations who have
offered assistance, guidance, and encouragement throughout this work.

Additional information
Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

MycoKeys 121: 79-92 (2025), DOI: 10.3897/mycokeys.121.158055 88

Muhammad Binyamin Khan et al.: Two new species of Meliola from Yunnan Province China

Use of Al

No use of Al was reported.

Funding

This study was supported by the National Natural Science Foundation of China (No.
32060012 and 32400013) and the Science and Technology Foundation of Guizhou Prov-
ince (No. [2019]2451-3). Their continued support and commitment to scientific prog-
ress have been invaluable throughout this study.

Author contributions

All authors have contributed equally.

Author ORCIDs

Muhammad Binyamin Khan © https://orcid.org/0000-0001-5979-1036
Xiang-Yu Zeng © https://orcid.org/0000-0003-1341-1004

Entaj Tarafder © https://orcid.org/0000-0002-3680-3433

Sardar Ali © https://orcid.org/0009-0006-7938-882X

DE-Ping Wei © https://orcid.org/0000-0002-3740-0142

Ting Chi Wen © https://orcid.org/0000-0003-1744-5869

Data availability

All of the data that support the findings of this study are available in the main text.

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